1. Field of the Invention
This invention relates to a polypeptide having thermally-tolerant pectin methylesterase activity, a gene encoding the polypeptide, a method of producing the polypeptide, a process for isolating the polypeptide, compositions comprising the polypeptide, and methods of using the polypeptide and compositions.
2. Description of the Relevant Art
Processed citrus juice from Florida (predominantly from oranges) is currently valued as an approximately $2.86 billion industry (Hodges et al. 2006. EDIS Document FE633, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Fla.). Product quality is largely defined by juice cloud properties such as turbidity, flavor, aroma, and color (Baker and Cameron. 1999. Food Technol. 53: 64-69). Cloud loss is a defect largely attributable to the enzyme pectin methylesterase (PME) (Rouse, A. H. 1949. Proc. Florida State Hort. Soc. 62: 170-173). PME acts on pectin associated with juice cloud, leading to aggregation through calcium cross-linking and subsequent cloud separation (Baker and Cameron, supra; Croak and Corredig. 2006. Food Hydrocolloids 20: 961-965). Multiple forms of PME are present in juice, but only certain PME forms are responsible for juice cloud separation (Versteeg et al. 1980. J. Food Sci. 45: 969-971, 998; MacDonald et al. 1993. J. Sci. Food Agric. 62: 163-168; Cameron et al. 1998. J. Food Sci. 63: 253-256). Variance in action on pectin may represent differences in mechanism of action by PME isoforms.
Citrus juice peel contains 10-15% pectin on a fresh weight basis (Grohmann and Baldwin. 1992. Biotechnol. Left. 14: 1169-1174; Grohmann et al. 1995. Biores. Technol. 54: 129-141). Pectin, a complex polysaccharide, is composed of at least five different sugar moieties, but 80-90% of its dry weight is galacturonic acid. The vast majority of the galacturonic acid is found in homogalacturonan regions of pectin, regions of unbranched polymers of galacturonic acid in which a variable proportion of the galacturonic acid residues may contain a methyl ester at their C6 position. The functional properties of pectin are thought to be dependent on the fraction of these galacturonic acid residues that are methylated and their distribution along the homogalacturonan stretches (Taylor, A. J. 1982. Carbohydrate Polymers 2: 9-17; Willats et al. 2001. J. Biol. Chem. 276: 19494-19413). Two patterns of methyl ester distribution are generally recognized, either random or ordered, i.e., blockwise (Willats et al., supra). Analyzing these patterns of demethylation is key to understanding their relationship to function.
Citrus juice cloud stability and the functional properties of commercial citrus pectin are related to the pectin molecular weight (Hotchkiss et al. 2002. J. Agric. Food Chem. 50: 2931-2937), degree of methylesterification (Hills et al., 1949. Food Technol. 3: 90-94), and intramolecular distribution of the methyl ester blocks within the population of pectin molecules (Joye and Luzio. 2000. Carbohydr. Polym. 43: 337-342; Willats et al., supra; Baker, R. A. 1979. J. Agric. Food Chem. 27:1387-1389; Wicker et al. 2003. Food Hydrocolloids 17: 809-814). Sequential cleavage of the C6 methyl esters of galacturonic acid residues in pectin produces a distribution of “blocks” of free acid groups. When of sufficient size, such blocks on adjacent pectin molecules can be cooperatively cross-linked by divalent cations, leading to precipitation of the pectins. In the presence of juice cloud and adequate serum calcium ions, this precipitation entrains and removes the cloud particulates resulting in an unattractive, largely flavorless clear serum, deficient in sensory properties (Cameron et al. 1998, supra). Although a minimal block size of nine unesterified galacturonic acid residues has been hypothesized to be necessary for calcium cross-linking, a larger de-esterified block might be necessary for gel formation (Liners et al. 1992. Plant Physiol. 99: 1099-1104; Corredig et al. 2001. J. Agric. Food Chem. 49: 2523-2526). Gelation in juice concentrates may vary from slight curdiness to firm gels, which are unsightly and can hinder reconstitution (Cameron et al. 1998, supra). Approximately 25-20% of cloud-associated pectin has been reported to be calcium pectate (Klavons et al. 1994. J. Food Sci. 59: 399-401). High-methoxy pectins in which de-esterified blocks allow them to gel in the presence of calcium without the addition of sucrose are termed calcium-sensitive pectins (Hills et al., supra; Joye and Luzio, supra). Two thermally labile pectin methylesterases (TL-PMEs) from citrus fruit pulp tissue have been demonstrated to be blockwise demethylating enzymes, introducing calcium sensitivity into a non-calcium-sensitive pectin (NCSP) with very limited reduction (6.0%-6.5%) in their degree of methylesterification (Savary et al. 2001. J. Agric. Food Chem. 50: 3553-3558; Cameron et al. 2001. J. Agric. Food Chem. 51: 2070-2075).
Pectin methylesterases (PMEs) are cell wall polysaccharide-modifying enzymes (EC 3.1.1.11) that act to hydrolyze the C6-carboxyl methyl esters decorating homo-galacturonan regions of pectin described above (Bordenave, M. 1996. In: Plant Cell Wall Analysis, Linskens and Jackson, eds., Springer-Verlag, New York, pp. 165-180; Micheli, F. 2001. Trends Plant Sci. 6: 414-419; Markov{hacek over (c)} and Jane{hacek over (c)}ek. 2004. Carbohyd. Res. 339: 2281-2295). Thus, control of these enzyme activities in juice processing from fruit species such as Citrus is critical for quality and stability of fruit juices (Klavons et al., supra; Goodner et al. 1998. J. Agric. Food. Chem. 46: 1997-2000; Baker and Cameron, supra). PME action to de-esterify pectin in juice cloud colloidal structures results in calcium-mediated agglomeration and subsequent irreversible cloud separation, thus yielding degraded juice. Citrus species produce multiple forms of PMEs in fruit tissues (Versteeg et al. 1980, supra; Seymour et al. 1991a. J. Agric. Food. Chem. 39: 1080-1085; Seymour et al. 1991b. J. Agric. Food. Chem. 39:1075-1079; MacDonald et al., supra; Cameron et al. 1998, supra.
PMEs are present as a large multi-gene family in plants (Micheli, supra; Markovi{hacek over (c)} and Jane{hacek over (c)}ek, supra; Pelloux et al. 2007. Trends Plant. Sci., 12: 267-277). They appear to represent the largest cell wall-related gene families in both Arabidopsis and Oryza (Yokoyama and Nishitani. 2004. Plant Cell Physiol. 45: 111.1-1121). As many as seven unique nucleic acid sequences have been obtained from orange and reported to public sequence databases, and their sequence similarities indicate they represent variants of the two major TL-PMEs (Nairn et al. 1998. Physiol. Plant 102: 226-235; Christensen et al. 1998. Planta 206:493-503; Arias and Burns. 2002. J. Agric. Food. Chem. 50: 3465-3472). Other partial PME-like sequences are present in Citrus sinensis EST sequence libraries (Bausher et al. 2003. Plant Sci. 165: 415-422; Savary et al. 2003. In: Advances in Pectin and Pectinase Research, Voragen et al., eds., Kluwer Academic Publishers, Netherlands, pp. 345-361). Some PMEs and their genes have been isolated from plants, bacteria and fungi and have been patented for use in modifying food hydrocolloids (Christensen et al. 2001 U.S. Pat. No. 6,268,195 Christensen et al. 2003 U.S. Pat. No. 6,627,429; Andersen et al. 2000. U.S. Pat. No. 6,069,000).
At the protein level, only the two major thermally-labile orange PMEs (TL-PME) have been purified to homogeneity to provide amino acid sequence information in conjunction with biochemical properties. The two PMEs are represented by what have been described as salt-independent peak 2 PME, PME2 (Savary et al. 2002. J. Agric. Food. Chem. 50: 3553-3358) and salt-dependent peak 4 PME, PME4 (Cameron et al. 2003. J. Agric. Food Chem. 51: 2070-2075). In Valencia orange, the most abundant PME form present in pulp tissue, the thermolabile salt-independent PME2, readily destabilizes juice at room temperature (Cameron et al. 1998, supra; Savary et al. 2002, supra).
However, it is the thermally tolerant PME (TT-PME) that it is most significant in destabilizing juice cloud under cold storage at 4° C. (Versteeg et al., 1980, supra; Cameron et al.,1998, supra). TT-PMEs have been found in lemon juice, grapefruit pulp, citrus tissue culture cells, commercial fresh frozen Valencia orange juice, and other sweet orange varieties (MacDonald et al., supra; Seymour et al., supra; Cameron and Grohmann. 1995. J. Food Sci. 60: 821-825; Cameron et al. 1994. J. Agric. Food Chem. 42: 903-908; Cameron et al. 1996. J. Food Sci. 62: 242-245; Snir et al. 1996. J. Food Sci. 61: 379-382). TT-PME is found in less abundance than the TL-PMEs; however, its considerable tolerance to heat inactivation necessitates that it be strictly controlled during processing in order to stabilize juice cloud (Cameron and Grohmann. 1996. J. Agric. Food. Chem. 44: 458-462; Baker and Cameron, supra). Pasteurization of juice at approximately 20° C. above that temperature considered necessary to control microbial growth is required to effectively inactivate TT-PME (Eagerman and Rouse. 1976. J. Food Sci. 41: 1396-1397; Chen et al., 1998. J. Agric. Food Chem. 46: 1777-1782).
There is a problem of continuing uncertainty in the food biochemistry literature regarding PME behavior in juice and in vitro systems due to inadequate or ambiguous identification of the protein isoforms present in the preparations that have been studied. This is a critical issue, since various PME isoforms are known to be present in the same preparation and do not have identical biochemical properties and their action on pectin and ability to de-stabilize juice cloud vary (Versteeg et al., 1978. Lebensm-Wiss. Technol. 11: 267-274; MacDonald et al., 1996; Cameron et al., 1998, supra; Catoire et al. 1998. J. Biol. Chem. 273: 3310-3315; Corredig and Wicker. 2002. J. Food Sci. 67: 1668-1671).
To address isoform identification, we applied matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) and associated proteomics tools as a direct means to readily identify the two major orange TL-PMEs by matching tryptic peptides to their corresponding translated nucleotide sequences (Savary et al., 2007). Coupling this analytical technology with homogeneous protein preparations resulted in unequivocal structural evidence that the salt-independent TL-PME2 is encoded by the gene deduced from the sequence of the ˜36 kDa protein that Arias and Burns (supra) identified as associated with thermostable PME activity.
TT-PME (peak 3 PME, Cameron et al., 1998, supra) has been reported to be of higher molecular weight than the TL-PME isoforms, with sizes estimated as 40,000-43,000 to 51,000-53,000 (Versteeg et al. 1980, supra; Seymour et al., supra; Cameron and Grohman, 1996, supra; Cameron et al. 2005. J. Agric. Food Chem. 53:2255-2260). We had previously correlated TT-PME activity to a 41,000 glycoprotein by denaturing PAGE (Cameron et al, 1995; Cameron et al., 2005, supra). However, the N-terminal peptide sequences and MALDI-TOF MS spectra obtained from this 41,000 glycoprotein suggested identity to polygalacturonase inhibitor proteins (PGIPs). PGIPs are monomeric cell wall-associated proteins in citrus fruit regarded as defensive proteins that act to modify action of fungal pathogen polygalacturonases (D'hallewin et al. 2004. Physiol. Plant 120:395-404; Kemp et al. 2004. Plant-Microbe Interact. 17: 888-894; Federici et al. 2006. Trends Plant Sci. 11:65-70). A dual-functional protein, containing both PME and PGIP activities, would be remarkable but not unprecedented (Sharma et al. 2004. Plant Physiol. 134: 171-181; Roopashree et al. 2006. Biochem. J. 395: 629-639). If both activities are indeed present in a single protein, possession of a methylesterase activity would suggest a mechanism for how certain PGIPs may activate some fungal polygalacturonases (Kemp et al., supra). However, we were unable to either confirm dual activity for orange PGIP or demonstrate co-purification of a more abundant second protein—a PGIP with nearly identical physicochemical properties—with the protein responsible for TT-PME activity; our efforts to confirm identification were hampered by insufficient quantities of protein (Cameron et al., 2005, supra).
TT-PME activity is present as a small portion of total extractable PME activity in Citrus fruit (Seymour et al., supra; Snir et al, supra; MacDonald et al. 1994. J. Sc. Food Agric. 64: 129-134; Cameron et al., 1998, supra). Purification of TT-PME is further hampered by the presence of soluble pectinates co-extracted during PME isolation (Versteeg et al. 1980, supra; Snir et al., 1995; MacDonald et al., 1996; Cameron et al., 1998, supra). This has led to complex purification schemes, insoluble precipitates, and low yields. Given the difficulties presented by the low amounts of TT-PME activity measured and the presence of pectinates, previous experimental preparations could not conclusively associate TT-PME activity in the juice cloud with a protein having a definitive size, structure, sequence, and identifying biochemical characteristics.
Thus, TT-PME is a key fruit cell wall enzyme. There exists a need to improve the strategy for purifying orange TT-PME, and thereby enabling the isolation of a electrophoretically pure protein and establishing its unambiguous identification as a TT-PME with regard to its size, structure, sequence, and characteristic activity. We have developed a simplified purification method that provides greatly improved yield and purity of a TT-PME. The enzyme thus prepared was used to establish the identity of a novel TT-PME by biochemical assays, MALDI-TOF MS sequencing, and determination of its complete nucleotide sequence.